Increased parasite burden is a key factor in the severity of clinical toxoplasmosis and thus, pathogenesis in Toxoplasma gondii infections is caused primarily by the growth of the parasite. From recent genetic analysis it has become clear that a shortened tachyzoite cell cycle is a key virulence determinant in Toxoplasma, yet we have few molecular details about how replication is regulated in this parasite. Toxoplasma tachyzoites divide by a novel cycle where the duplication of a complex set of organelles is coordinated with an unusual bimodal S phase and a phylum-specific budding process is synchronized with, and may regulate, aspects of mitosis. The binary division of Toxoplasma tachyzoites undergoing endodyogeny offers advantages for the investigation of the apicomplexan cell cycle, although these studies will apply broadly to the growth of other pathogens in this family, such as Plasmodium, Eimeria, and Cryptosporidium, where our knowledge of parasite cell cycle mechanisms is equally deficient. In this application, we propose a comprehensive study of the mechanisms controlling the replication of virulent Type I-RH tachyzoites. In Aim 1, we will test the hypothesis that at least four checkpoints in G1, early and late S, and mitosis regulate the RH tachyzoite cell cycle through the analysis of a large collection of temperature sensitive (ts) growth mutants (165 total), which we have produced by chemical mutagenesis. In Aim 2, we will examine the hypothesis that known as well as unique apicomplexan proteins are required for tachyzoite checkpoint control through cosmid-based genetic complementation, which will identify the essential genes involved in specific ts-mutants. Finally, in Aim 3, we will define the role of the daughter/mitotic cytoskeletons in regulating checkpoints that control chromosome replication. In preliminary studies, we have established high throughput protocols for producing and analyzing the phenotype of cell cycle mutants and we have demonstrated robust new cosmid-based methods for genetic complementation in this parasite. These studies will provide insight into the mechanisms regulating parasite division and provide new targets upon which to disrupt parasite proliferation.